| As an important native resource insect,the Chinese honeybee(Apis cerana cerana)plays important ecological roles in pollinating the plants that bloom at low temperatures in early winter in China,and the pollination behavior of the honeybee is closely related to its olfactory system.As the first participant of the olfactory system,three main types have been found,including:odorant-binding proteins(OBPs),chemosensory proteins(CSPs)and Niemann-Pick C2-type proteins(NPC2).Therefore,taking the response of the antennae of A.cerana cerana to chemical volatiles in Rhaphiolepis bibas at low temperature as a clue,to study the mechanism of the interaction between the olfactory receptor proteins and chemical semiochemical substances of A.cerana cerana,and for in-depth analysis of the olfactory perception mechanism of A.cerana cerana to Rhaphiolepis bibas aroma in early winter,it is of great theoretical significance to further evaluate the ecological value of A.cerana cerana on pollination of low temperature flowering plants.Therefore,this study firstly used insect chemical ecology technology to analyze the chemical constituents of Rhaphiolepis bibas aroma chemical volatiles that caused the olfactory response of A.cerana cerana.Then used transcriptome sequencing technology to study related physiological processes and signaling pathways,the differential genes of the antennae after high and low temperature treatments were analyzed,and the olfactory-related genes(such as Ac CSP1,Ac OBP2 and Ac NPC2family)of A.cerana cerana olfactory in response to low temperature were preliminarily screened.The binding properties of Ac CSP1 and Ac OBP2 proteins to the corresponding Rhaphiolepis bibas aroma chemical semiochemicals were clarified by molecular docking,site-directed mutagenesis and thermodynamic techniques.law of action.Finally,in this study,the full length of the Ac NPC2 family was cloned for the first time and its spatiotemporal expression profile during the development of A.cerana cerana was preliminarily determined.This provides a theoretical basis for the in-depth understanding of the olfactory system of A.cerana cerana to specifically perceive the volatiles of pollinating plants at low temperature.The main results of this study are as follows:1.Combined GC-MS and GC-EAD techniques to analyze the chemical volatiles of the Rhaphiolepis bibas in the antennae of A.cerana cerana,and 9 species of chemical information components that responded to the antennae of A.cerana cerana were identified.The chemical volatiles of these 9 kinds of Rhaphiolepis bibas were verified at two temperatures of 12°C and 25°C.The results showed that although the low temperature response was slightly decreased,the antennae of A.cerana cerana showed the strongest olfactory responses to three volatiles,including(2-Nitroethyl)benzene,Methyl 4-methoxybenzoate and 4-methoxybenzoate.In addition,4 volatiles were significantly enhanced at low temperature and low concentration(0.1 m M),among which the Phenylethyl alcohol showed a significant difference(p<0.05)and the 4-Methoxybenzaldehyde showed a extremely significant difference(p<0.01),indicating that the two were involved in the process of low temperature olfactory perception of loquat flowers by A.cerana cerana.2.Transcriptome sequencing was performed on the antennae of A.cerana cerana harvester after low temperature treatment and after normal temperature capture.A total of 111 differentially expressed genes were identified in the annotated unigenes,and The 9 genes in the transcriptome sequencing results were verified by q PCR,and olfactory-related genes that responded to low temperature were screened,including the Ac CSP1,Ac OBP2 and Ac NPC2 family.The 9 differential genes in the transcriptome sequencing results were verified by q PCR,and the expression trend was consistent with the transcriptome sequencing results,indicating the reliability of the transcriptome sequencing results.Through GO and KEGG enrichment analysis,it was found that the differentially expressed genes were mainly enriched in GO items such as biological process,molecular function and cell membrane components,and were associated with tyrosine metabolism,linoleic acid metabolism,α-linolenic acid metabolism and ether lipid metabolism.related to multiple pathways.Functional analysis shows that low temperature affects multiple physiological processes and metabolic pathways in A.cerana cerana,enabling it to continue to visit flowers in a relatively low temperature environment,playing a more critical role than other insects.3.The prokaryotic expression and protein purification of the chemosensory protein Ac CSP1 and the odorant-binding protein Ac OBP2,which are related to the low temperature sensing of Rhaphiolepis bibas volatiles in A.cerana cerana.The results of the fluorescence competition binding experiment showed that among all the9 chemical volatiles of Rhaphiolepis bibas that caused the antennal response of A.cerana cerana,Ac CSP1 could compete with the relative fluorescence value of the fluorescent reporter 1-NPN to less than 50%,while Ac OBP2 had only five species,and Ac CSP1 has a stronger affinity than Ac OBP2.From the K_D value of the dissociation constant,4-methoxybenzaldehyde,Methyl cinnamate and(E)-Ethyl cinnamate all showed stronger binding ability with them.For Ac CSP1,the dissociation constant K_D values of these three chemical semiochemicals are 2.07μmol/L,3.22μmol/L and 3.03μmol/L,respectively.For Ac OBP2,the dissociation constant K_D values are 7.46μmol/L,11.54μmol/L and 9.63μmol/L.4.Determination of key amino acid sites,interaction force and point mutation experiments of Ac CSP1 and Ac OBP2 binding to ligands.Fluorescence quenching and thermodynamic analysis were performed on the mutant proteins(Ac CSP1-F44G and Ac CSP1-Q63G,Ac OBP2-M55G and Ac OBP2-K51G),the K_A values of the mutant proteins and the corresponding ligands were decreased,and the binding force was weakened.It seems that F44 may play a greater role in the binding of Ac CSP1 to4-methoxybenzaldehyde,Methyl cinnamate,and(E)-Ethyl cinnamate.In the binding of Ac OBP2 to 4-methoxybenzaldehyde M55 plays a greater role in Ac OBP2.K51plays a greater role in the binding of Ac OBP2 to methyl and(E)-Ethyl cinnamate.The results showed that F44 and Q63 maybe two important amino acid residues to maintain the hydrophobic interaction and electrostatic force between Ac CSP1 and4-methoxybenzaldehyde,and the hydrophobic interaction with methyl cinnamate.An important amino acid residue in the electrostatic force between methyl cinnamate.K51 may be an important amino acid residue for maintaining the hydrophobic interaction and electrostatic force between Ac OBP2 and Methyl cinnamate.5.Clone and analyze the structural characteristics and expression profiles of the Ac NPC2 family(Ac NPC2a,Ac NPC2b,Ac NPC2c,Ac NPC2d),and analyze the differences in expression levels under high and low temperature treatments.The full length ORFs of 4 Ac NPC2 genes were obtained as 447,480,459,and 465 bp,respectively,encoding 148,159,152,and 154 amino acids.The predicted protein molecular weight is 16-19 k Da,and the isoelectric points are 7.98,7.57,6.56,and6.34,respectively.Phylogenetic tree analysis showed that Ac NPC2 sequences were most close to the NPC2 homologous sequence of Apis mellifera.q RT-PCR results showed that the expression level of Ac NPC2a was the highest in the abdomen of the newborns,followed by the abdomen of the nurses and the larval stage.The expression of Ac NPC2b was the highest in the chest of the newborn bees,followed by in the head,thorax and metapodium of the foragers.Ac NPC2 c was notably expressed in high abundance in the antennae of the nurses and the foragers.Ac NPC2d had the highest expression in the head of the foragers.Interestingly,after low temperature treatment,although there was no significant difference,all the expression levels of all Ac NPC2genes in the forager antennae increased,indicating that this gene family may be involved in the low temperature adaptation of A.cerana cerana,or related to flower visiting behavior in early winter. |
PDF Full Text Request